Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Biochemistry. 2010 Sep 28;49(38):8425-33. doi: 10.1021/bi101035k.
A typical feature of type II restriction endonucleases (REases) is their obligate sequence specificity and requirement for Mg(2+) during catalysis. R.KpnI is an exception. Unlike most other type II REases, the active site of this enzyme can accommodate Mg(2+), Mn(2+), Ca(2+), or Zn(2+) and cleave DNA. The enzyme belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. Residues D148, H149, and Q175 together form the HNH active site and are essential for Mg(2+) binding and catalysis. The unique ability of the enzyme to cleave DNA in the presence of different metal ions is exploited to generate mutants that are specific to one particular metal ion. We describe the generation of a Mn(2+)-dependent sequence specific endonuclease, defective in DNA cleavage with Mg(2+) and other divalent metal ions. In the engineered mutant, only Mn(2+) is selectively bound at the active site, imparting Mn(2+)-mediated cleavage. The mutant is impaired in concerted double-stranded DNA cleavage, leading to accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altered reaction conditions. The active site fluidity of REases allowing flexible accommodation of catalytic cofactors thus forms a basis for engineering selective metal ion-dependent REase additionally possessing nicking activity.
Ⅱ型限制内切酶（REases）的一个典型特征是其必需的序列特异性和催化过程中对 Mg2+的需求。R.KpnI 是一个例外。与大多数其他Ⅱ型 REases 不同，该酶的活性位点可以容纳 Mg2+、Mn2+、Ca2+或 Zn2+并切割 DNA。该酶属于 HNH 核酸酶超家族，其特征是存在 ββα-Me 指模体。残基 D148、H149 和 Q175 共同构成 HNH 活性位点，对于 Mg2+结合和催化至关重要。该酶在不同金属离子存在下切割 DNA 的独特能力被利用来产生对特定金属离子具有特异性的突变体。我们描述了一种 Mn2+依赖性序列特异性内切酶的产生，该酶在 Mg2+和其他二价金属离子存在下不能切割 DNA。在工程突变体中，只有 Mn2+被选择性地结合在活性位点上，赋予 Mn2+介导的切割。该突变体在协同双链 DNA 切割方面存在缺陷，导致切口中间产物的积累。通过改变反应条件，进一步增强了突变酶的切口活性。允许催化辅因子灵活适应的 REases 活性位点的流动性，从而为工程化选择性金属离子依赖型 REase 提供了基础，该酶还具有切口活性。
